Monoclonal antibody for detecting aflatoxin, ELISA (enzyme-linked immunosorbent assay) method and kit

An aflatoxin and monoclonal antibody technology, which is applied in the field of veterinary drug residue analysis and immunology, can solve the problems of inconvenient grass-roots use, low antibody sensitivity, complicated processing process, etc., and achieves low detection cost, good precision, and short cycle time. Effect

Active Publication Date: 2016-04-27
HUAZHONG AGRI UNIV
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  • Description
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AI Technical Summary

Problems solved by technology

Wang Xiong et al. (2008) used the ELISA method to detect aflatoxin B1 in feed. The minimum detection limit of the antibody was 0.05ng / mL, and the linear range of the standard curve was 0.05-2.00ng / mL. The sensitivity of the antibody was not high, and the use of The sample pretreatment process is relatively complicated, and it is not convenient to use at the grassroots level; Wang Lei et al. (2012) prepared an anti-aflatoxin B1 antibody, and the IC50 value of the antibody was 2.58ng / mL; Li Min et al. (2015) used heterologous coating The ELISA method was established for the antigen, and the sensitivity of the antibody was increased from 1.6ng / mL to 0.3ng / mL, and the ELISA method was successfully established

Method used

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  • Monoclonal antibody for detecting aflatoxin, ELISA (enzyme-linked immunosorbent assay) method and kit
  • Monoclonal antibody for detecting aflatoxin, ELISA (enzyme-linked immunosorbent assay) method and kit
  • Monoclonal antibody for detecting aflatoxin, ELISA (enzyme-linked immunosorbent assay) method and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] The preparation of embodiment 1 immunogen and coating former

[0027] 1.1 Preparation of immunogen AFB2O-DCC-KLH

[0028] Take 2mL of AFB2 methanol solution with a concentration of 1mg / mL, add 3mg of carboxymethylhydroxylamine hemihydrochloride (CMO) and 5mg of sodium carbonate, stir and react at room temperature for 24h, then blow dry with nitrogen, add 0.05mol / L 2 mL of hydrochloric acid, fully dissolve it, add 2 mL of ethyl acetate for extraction, take the ethyl acetate layer, and repeat 3 times; dry the obtained liquid under nitrogen to obtain the hapten aflatoxin B2 oxime (AFB2O).

[0029] Dissolve the obtained hapten with DMF, add 12 mg of DCC and 5 mg of NHS, and activate for 12 hours to obtain solution A; dissolve 2 mL of KLH in 5 mL of PBS solution to obtain solution B. Then slowly add solution A into solution B, and react for 12 hours under ice bath conditions; after the reaction is complete, put it into a dialysis bag, dialyze in PBS solution for 3 to 5 days...

Embodiment 2

[0032] The preparation of embodiment 2 monoclonal antibody

[0033] 2.1 Animal immunity

[0034] Balb / C mice (purchased from the Experimental Animal Center of Hubei Academy of Medical Sciences) were immunized with the immunogen (AFB2O-DCC-KLH) prepared by the inventor's National Laboratory for Veterinary Drug Residues. The immunization procedure is to take a protein solution containing 50-80 μg of AFB2O-DCC-KLH conjugate, mix it with an equal amount of adjuvant, and then inject it into the mouse body to make it produce specific serum.

[0035] 2.2 Cell fusion and cloning

[0036] With reference to Yang Hanchun's "Animal Immunology", the AFB2O-DCC-KLH immunogen prepared by the inventor's National Laboratory for Veterinary Drug Residues was used to immunize Balb / C mice (purchased from the Experimental Animal Center of the Hubei Provincial Center for Disease Control and Prevention), and the immunization procedure was as follows: : For basic immunization, the immunogen was emuls...

Embodiment 3

[0041] Example 3 Establishment of AFB1 Indirect Competitive ELISA Detection Method

[0042] 3.1 Preparation of reagents (the reagents used in this example were prepared by the following methods unless otherwise specified)

[0043] Phosphate buffer: NaCl8.0g, KH 2 PO 4 0.2g, Na 2 HPO 4 12H 2 O2.9g, KCl0.2g, add double distilled water to 1000mL, adjust pH to 7.4;

[0044] Coating solution: Take Na 2 CO 3 1.59g, NaHCO 3 2.93g, add triple distilled water to 1000mL, adjust the pH value to 9.6;

[0045] Washing solution: NaCl8.0g, KH 2 PO 4 0.2g, Na 2 HPO 4 12H 2 O2.9g, KCl0.2g, Tween200.5mL, add double distilled water to 1000mL, adjust pH to 7.4;

[0046] Blocking solution: 1.5g skimmed milk powder dissolved in 100mL phosphate buffer;

[0047] Substrate solution A: 3,3',5',5-tetramethylbenzidinediamine (TMB) 200mg, absolute ethanol 100mL, add double distilled water to 1000mL;

[0048] Substrate B: Na 2 HPO 4 14.6g, citric acid 9.3g, 0.75% urea hydrogen peroxide 6....

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Abstract

The invention discloses a specific monoclonal antibody capable of recognizing aflatoxin B1, B2, G1 and G2. The monoclonal antibody is secreted by a hybridoma cell strain AFB1 / 3B9, the hybridoma cell strain AFB1 / 3B9 is preserved in the CCTCC (China Center for Type Culture Collection), and the collection number is CCTCC NO: C201558. The invention further discloses an ELISA (enzyme-linked immunosorbent assay) method for detecting aflatoxin B1, B2, G1 and G2 and a kit. Compared with the prior art, the prepared monoclonal antibody can recognize aflatoxin B1, B2, G1 and G2 simultaneously and has very high recognition sensitivity; the ELISA and the kit have the advantages of simplicity, convenience, quickness, sensitivity, accuracy and the like.

Description

technical field [0001] The invention belongs to the technical field of veterinary drug residue analysis and immunology, and specifically relates to a monoclonal antibody capable of recognizing aflatoxin B1, B2, G1, G2 and an enzyme for detecting aflatoxin B1, B2, G1, G2 Linked immunoassay (ELISA) and kits. Background technique [0002] Aflatoxins are mainly secondary metabolites secreted by Aspergillus flavus and Aspergillus parasiticus. They are a class of compounds with similar structures. They all contain difuran rings and coumarins. There are more than 20 kinds of them. Currently, 12 kinds have been isolated and identified. The main ones are aflatoxin B1, B2, G1, G2, M1, M2. Aflatoxins mainly contaminate crops and feed. The use of feed contaminated with aflatoxins can cause stillbirths and mummified fetuses in pigs, and can cause decreased egg production and oral ulcers in chickens. Research has found that aflatoxin has obvious "three-cause" effects, and people who ea...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/14C12N5/20G01N33/577C12R1/91
CPCC07K16/14G01N33/577G01N2430/00
Inventor 袁宗辉陶燕飞彭大鹏杨碧嘉王玉莲潘源虎陈冬梅
Owner HUAZHONG AGRI UNIV
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